Selective peptide bond cleavage with chemical reagents has proven to be of immeasurable value in the determination of amino acid sequence. Methionine is the most utilized amino acid site of cleavage due to the exquisite action of cyanogen bromide. The cleavage target of second preference is tryptophan, for which a variety of reagents have been described. Despite more than twenty-five years of investigation, none of the tryptophan reagents can routinely match the cleavage efficiency and selectivity achievable at methionine.
Methods in selective protein cleavage have assumed an increased level of importance with the advent of protein semisynthesis and genetically-engineered protein synthesis. In these areas, utilization of cyanogen bromide has been nearly exclusive. The use of cyanogen bromide for synthetic purposes is precluded when methionine is an inherent residue of a peptide. Several peptides, in particular atrial naturietic factor (ANF), growth hormone releasing factor (GRF), and insulin-like growth factor-I (IGF-I) possess a single methionine and no tryptophan. Each peptide requires a reliable synthetic source for determination of its physiological significance and clinical utility. Currently, the most high-yielding production of peptides in this molecular size is through selective cleavage of an E. coli-synthesized fusion protein.
Bacterial synthesis of a fusion protein in which tryptophan immediately precedes a natural peptide devoid of it provides a potential site for selective cleavage. Chemical cleavage at tryptophan peptide bonds is achieved through oxidative halogenation, and has been extensively reviewed. [Fontana, A., Savige, W. E., and Zambonin, M. (1980) In "Methods in Peptide and Protein Sequence Analysis", (Birr, C., Ed.) Elsevier/North-Holland Biomedical Press, 309-322]. Cleavage yields approaching 60% have been attained through the action of BNPS-skatole [2-(2-nitrophenyl-sulfenyl)-3-methyl-3-bromoindolene]. More recently, selective cleavage by a mixture of DMSO and HBr in acetic acid has been recommended [Savige, W. E., and Fontana, A. (1977) Methods Enzymol. 47, 459-469]. Modification at tryptophan is not selective by either of these methods. Formation of methionine sulfoxide can occur at near quantitative levels. To a lesser extent, irreversibly modified amino acids such as methionine sulfone, cysteic acid, and/or brominated tyrosine have been observed.
The degree of side-reactions which occur in the course of tryptophan cleavage has been reported to be minimized with the reagent N-chlorosuccinimide (NCS) [Shechter, Y., Patchornik, A., and Burstein, Y. (1976) Biochem. 15, 5071-5075]. Methionine conversion to its sulfoxide was the only other modification originally detected. However, in a subsequent study with NCS, appreciable levels of cysteic acid and methionine sulfone were observed [Lischwe, M. A., and Sung, M. T. (1977) J. Biol. Chem. 252, 4976-4980]. In amino acid sequence determination, these side-reactions can be distracting but do not preclude successful utilization [Huang, H. V., Bond, M. W., Hunkapillar, M. W., and Hood, L. E. (1983) Methods Enzymol. 91, 318-324]. More stringent requirements apply to the use of these reagents during the course of protein synthesis.
Conceptually, tryptophan cleavage with DMSO and HBr in acetic acid is most attractive. Cleavage is selective with reagents that are inexpensive and readily available. More importantly, there is the potential opportunity for immediate regeneration of methionine. Reduction of methionine sulfoxide is achievable in concentrated hydrochloric acid through the addition of dimethyl sulfide [Savige, W. E., and Fontana, A. (1977) Methods Enzymol. 47, 453-459]. However, due to the deleterious effects of the strong acidic conditions on peptide structure, these approaches to tryptophan cleavage and methionine sulfoxide reduction are rarely used. In principle, if an appropriate solvent were identified, DMSO-promoted tryptophan cleavage could be rapidly followed by DMS-induced methionine regeneration.
Efficient cleavage at tryptophan cannot be achieved with DMSO and 4N hydrochloric acid in acetic acid [Savige, W. E., and Fontana, A. (1977) Methods Enzymol. 47, 442-453]. The action of a more potent halogenating agent such as HBr is required. Unfortunately, its presence also results in an undesirable increase in the level of cysteic acid.
Thus, it is of great importance to develop a process which will afford efficient cleavage at tryptophan while minimizing formation of undesirable by-products and especially by-products that are irreversible.
Such a process has now been discovered. It retains the use, as in prior art processes, of DMSO and HCl. However, instead of carrying out the cleavage in acetic acid, trifluoroacetic acid is employed. Substantial levels of tryptophan cleavage occur but with minimal production of irreversible side products, in particular, minimal conversion of cysteine to cysteic acid and of methionine to methionine sulfone.